Genetic Regulatory Elements and Uses Thereof

This application claims priority to U.S. Provisional Patent Application No. 63/354,110, filed Jun. 21, 2022, which is incorporated by reference herein in its entirety for all purposes.

The contents of the electronic sequence listing (REGT_002_01WO_SeqList_ST26.xml; Size: 31,745 bytes; and Date of Creation: Jun. 14, 2023) are herein incorporated by reference in their entirety).

The disclosure relates to genetic regulatory elements and uses thereof to express heterologous nucleotide sequences.

Gene therapy has the potential to revolutionize the treatment of genetic disorders. Clinical application of this promising technology requires overcoming significant technical limitations. Among these is the need to restrict the therapeutic intervention specifically to the cellular populations affected by the disease in order to reduce off-target effects and increase efficacy of the therapy.

Provided herein is a vector comprising (i) a heterologous nucleotide sequence; and (ii) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11. In some embodiments, the vector further comprises a promoter.

In some embodiments, the vector comprises, in 5′ to 3′ order: (i) the promoter; (ii) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (iii) the heterologous nucleotide sequence.

In some embodiments, the vector comprises, in 5′ to 3′ order: (i) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (ii) the promoter; and (iii) the heterologous nucleotide sequence.

In some embodiments, the vector comprises, in 5′ to 3′ order: (i) the promoter; (ii) the heterologous nucleotide sequence; and (iii) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid vector.

In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector or an adenovirus vector.

In some embodiments, the AAV vector comprises a 5′ AAV inverted terminal repeat (ITR) and a 3′ AAV ITR flanking the heterologous nucleotide sequence, a promoter, and (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′ AAV ITR; (ii) the promoter; (iii) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′ AAV ITR; (ii) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (iii) the promoter; (iv) the heterologous nucleotide sequence; and (v) the 3′ AAV ITR.

In some embodiments, the AAV vector comprises, in 5′ to 3′ order: (i) the 5′ AAV ITR; (ii) the promoter; (iii) the heterologous nucleotide sequence; (iv) (a) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (b) a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (c) a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; (d) a reverse complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or (e) a nucleotide sequence comprising one or more regions of about 100 bp or longer having at least 75% or greater sequence identity to any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; and (v) the 3′ AAV ITR.

In some embodiments, the promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE).

In some embodiments, the promoter is a minimal promoter. In some embodiments, the minimal promoter is a human beta-globin minimal promoter.

In some embodiments, the vector further comprises an enhancer. In some embodiments, the vector further comprises or encodes a polyadenylation signal sequence. In some embodiments, the vector further comprises or encodes a Kozak sequence. In some embodiments, the vector further comprises or encodes a woodchuck hepatitis virus post-transcriptional element (WPRE), a hepatitis B virus posttranscriptional regulatory element, an RNA transport element (RTE), a WPRE3, or a wsl3 regulatory element. In some embodiments, the vector further comprises an artificial intron. In some embodiments, the vector further encodes a 2A self-cleaving peptide. In some embodiments, the vector further comprises or encodes an internal ribosome entry site (IRES).

In some embodiments, the heterologous nucleotide sequence encodes a protein. In some embodiments, the heterologous nucleotide sequence encodes a gene silencing nucleic acid. In some embodiments, the heterologous nucleotide sequence encodes a CRISPR-associated protein. In some embodiments, the heterologous nucleotide sequence further encodes a guide RNA (gRNA).

Provided herein is a viral particle comprising a vector disclosed herein. In some embodiments, the viral particle is a recombinant adenovirus particle. In some embodiments, the viral particle is a recombinant AAV (rAAV) particle. In some embodiments, the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh.74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, TM-AAV6, AAV-PHP.A, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP.B10, AAV2-r3.45, AAV2-LSS, AAV2PFG, AAV2-PPS, AAV2-TLH or AAV2-GMN serotype particle.

Provided herein is a population of viral particles comprising a plurality of viral particles disclosed herein.

Provided herein is a pharmaceutical composition comprising a vector, a viral particle, or a population disclosed herein, and a pharmaceutically acceptable carrier, vehicle or diluent.

Provided herein is a nanoparticle comprising a vector disclosed herein.

Provided herein is a cell comprising a vector or a viral particle disclosed herein. In some embodiments, the cell is a mammalian cell or an insect cell.

Provided herein is a method of producing a rAAV particle, the method comprising: (i) culturing a cell disclosed herein under conditions allowing for packaging the rAAV particle; and (ii) harvesting the cultured host cell or culture medium for collection of the rAAV particle.

Provided herein is a method for selectively expressing the heterologous nucleotide sequence in neurons of the central nervous system (CNS) in a subject in need thereof, the method comprising administering to the subject a vector, a viral particle, a population, a pharmaceutical composition, or a nanoparticle disclosed herein.

In some embodiments of the methods provided herein, the heterologous nucleotide sequence expression is enriched in specific regions of the CNS. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of one or more of: the forebrain, the cerebellum, and the spinal cord. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the forebrain and the cerebellum. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the forebrain. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum and hippocampus. In some embodiments, the heterologous nucleotide sequence expression is enriched in neurons of the spinal cord.

In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO: 10, and the heterologous nucleotide sequence expression is enriched in neurons of the CNS.

In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 9, and the heterologous nucleotide sequence expression is enriched in neurons of the forebrain and the cerebellum.

In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 11, and the heterologous nucleotide sequence expression is enriched in neurons of the forebrain.

In some embodiments of the methods provided herein, the vector comprises the nucleotide sequence of SEQ ID NO: 6, and the heterologous nucleotide sequence expression is enriched in neurons of the cerebellum and hippocampus.

In some embodiments of the methods provided herein, the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject intravenously. In some embodiments of the methods provided herein, the vector, the viral particle, the population, the pharmaceutical composition, or the nanoparticle is administered to the subject via injection into the CNS.

The disclosure provides regulatory elements capable of selectively expressing a heterologous nucleotide sequence in specific neuronal populations and regions of the central nervous system (CNS) while reducing or preventing expression of the heterologous nucleotide sequence in other organs. The regulatory elements enrich expression of the heterologous nucleotide sequence in neurons and reduce or prevent expression in other cellular populations (e.g., glia, microglia, and endothelial cells). When integrated into a DNA-based expression vector, the regulatory elements can be used to reduce off-target effects and increase efficacy of gene therapy. The regulatory elements show evidence of broad applicability, as delivery of the same regulatory element with different rAAV serotypes, in differently aged mice, and expressing different transgenes demonstrated the same expression profile.

Provided herein are genetic regulatory elements for selectively expressing a heterologous nucleotide sequence in neurons of the CNS. When integrated in a DNA-based expression vector, these regulatory elements are capable of restricting the expression of the heterologous nucleotide sequence to neurons of the CNS. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the CNS with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the CNS with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the forebrain and cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the forebrain and cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the forebrain with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the forebrain with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the cerebellum with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the cerebellum and neurogenic niches with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% specificity for this cell type. In some embodiments, the regulatory elements restrict the expression of the heterologous nucleotide sequence to neurons of the cerebellum and neurogenic niches with at least about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% selectivity for this cell type.

In some embodiments, the regulatory elements express the heterologous nucleotide sequence in non-neuronal cells of the CNS (e.g., glial cells, microglial cells or endothelial cells) with at most about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% specificity for this cell type. In some embodiments, the regulatory elements express the heterologous nucleotide sequence in non-neuronal cells of the CNS (e.g., glial cells, microglial cells or endothelial cells) with at most about 25%, about 20%, about 15%, about 10%, about 5%, or about 1% selectivity for this cell type.

In some embodiments, the regulatory elements do not express the heterologous nucleotide sequence in one or more of liver, heart, muscle, and lung.

Provided herein is a genetic regulatory element comprising the nucleotide sequence of any of SEQ ID NOs: 1-13, a reverse nucleotide sequence of any of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any of SEQ ID NOs: 1-13; or a reverse complementary sequence of any of SEQ ID NOs: 1-13.

Provided herein is a genetic regulatory element comprising the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a reverse complementary sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

Provided herein is a vector (e.g., a DNA vector) comprising (i) a heterologous nucleotide sequence; and (ii) the nucleotide sequence of any of SEQ ID NOs: 1-13; a reverse nucleotide sequence of any of SEQ ID NOs: 1-13; a complementary nucleotide sequence of any of SEQ ID NOs: 1-13; or a reverse complementary sequence of any of SEQ ID NOs: 1-13.

Provided herein is a vector (e.g., a DNA vector) comprising (i) a heterologous nucleotide sequence; and (ii) the nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a reverse nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; a complementary nucleotide sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; or a reverse complementary sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

In some embodiments, a vector comprises (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer having at least 75%, 80%, 85%, 90%, 95%, 97%, or 99% or greater sequence identity to any of SEQ ID NOs: 1-13.

In some embodiments, a vector comprises (i) a heterologous nucleotide sequence; and (ii) a nucleotide sequence comprising one or more regions of about 100 base pairs (bp) or longer having at least 75%, 80%, 85%, 90%, 95%, 97%, or 99% or greater sequence identity to any of SEQ ID NOs: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11.

In some embodiments, a vector comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the regulatory elements disclosed herein. In some embodiments, a vector comprises 2, 3, 4, 5, 6, 7, or 8 of the regulatory elements CNS2_V4 (SEQ ID NO: 1), CNS2_V4.1 (SEQ ID NO: 10); CNS2_V3 (SEQ ID NO: 2), CNS2_V3.3 (SEQ ID NO: 9); CNS2_V7 (SEQ ID NO: 4), CNS2_V7.1 (SEQ ID NO: 11), CNS2_V11 (SEQ ID NO: 5), and CNS2_V10 (SEQ ID NO: 6).

In some embodiments, a vector provided herein further comprises a promoter. In some embodiments, the promoter is a minimal promoter. In some embodiments, a minimal promoter comprises one or more short sequences capable of promoting transcription. In some embodiments, a promoter comprises an RNA-polymerase II binding site, a TATA box, a TF2B recognition element (BRE), a motif 10 element (MTE), or a downstream promoter element (DPE). A TATA transcriptional regulatory activation site is described in e.g., François et al., (2005)79(17):11082-11094.

In some embodiments, the promoter is a human beta-globin minimal promoter.

In some embodiments, a promoter is recognized by RNA polymerase II. In some embodiments, a promoter is recognized by RNA polymerase III. In some embodiments, a promoter is a human U6 (hU6) promoter, a mouse U6 promoter or a human H1 promoter.

In some embodiments, a promoter is a constitutive promoter. In some embodiments, a promoter is an inducible promoter. In some embodiments, a promoter is a tissue-specific promoter. In some embodiments, a promoter is the chicken beta-actin (CBA) promoter, the GUSB240 promoter, the GUSB379 promoter, the HSVTK promoter, the CMV promoter, the SV40 early promoter, the SV40 late promoter, the metallothionein promoter, the murine mammary tumor virus (MMTV) promoter, the Rous sarcoma virus (RSV) promoter, the polyhedrin promoter, the EF-1 alpha promoter, the dihydrofolate reductase (DHFR) promoter or the phosphoglycerol kinase (PGK) promoters.